The objective of this proposal is to identify novel biomarkers and molecular mechanisms of multi-walled carbon nanotube (MWCNT)-induced pulmonary diseases, including fibrosis, for early detection and treatment interventions. MWCNT have been widely used for various industrial applications. However, concern over potential MWCNT-induced toxicity has emerged, particularly due to the structural similarity between asbestos and MWCNT. In our preliminary studies, exposure of mice to MWCNT by pharyngeal aspiration results in significant pulmonary inflammation, damage, and fibrosis at 56 days post-exposure. We have identified MWCNT-induced gene signatures in the mouse model that could predict human lung cancer risk and progression. In the computational evaluation of more than 800 pathways, VEGF, ICAM-1, MCP-1, and TGF- are among the most significantly represented pathways in the mouse model. The involvement of these signaling pathways has been further validated in MWCNT-treated human small airway epithelial cells (SAEC). A co-culture model of SAEC with human microvascular endothelial cells (HMVEC) was recently developed by our laboratory to elucidate mechanisms for MWCNT-induced cellular response. Nevertheless, the long-term pulmonary responses to MWCNT in mice and their underlying molecular mechanisms remain to be resolved. Moreover, there are currently no clinically available biomarkers for early detection and no effective treatment inventions for MWCNT-induced pulmonary diseases, particularly lung fibrosis. We hypothesize that genomic profiling and computational toxicology analysis of in vitro and in vivo studies can identify novel mechanisms and biomarkers predictive of MWCNT-induced pulmonary injuries in humans, which will lead to early diagnostic detection and treatment interventions for MWCNT-induced pulmonary diseases, including fibrosis. We will utilize multidisciplinary approaches, including in vivo animal toxicology assays, in vitro cellular toxicology assays, and computational modeling, to establish in vitro genomic signatures predictive of MWCNT-induced pulmonary diseases in the in vivo animal model and in humans and to explore the potential molecular targets for early treatment interventions. Aim 1 will evaluate the pulmonary dose-response and time course responses to MWCNT exposure in mice for up to 1 year. Aim 2 will determine the molecular mechanisms of MWCNT- induced injuries to the lung using an alveolo-capillary co-culture model. Aim 3 will identify mechanism-based gene signatures predictive of MWCNT-induced human pulmonary diseases using in vivo, in vitro, and patient data for early detection and treatment interventions. Aim 4 will perform integrated analyses of MWCNT-induced mRNA and miRNA changes and identify miRNA markers for early detection of MWCNT-induced lung fibrosis using non-invasive blood tests. We anticipate that this project will transform toxicological research of MWCNT- induced pulmonary diseases into strategies for environmental health protection and intervention. Results will fill th gap between in vivo/in vitro MWCNT-induced toxicity studies and risk assessment in humans.